A highly sensitive method for the determination of protein bound 3,4-dihydroxyphenylalanine as a marker for post-translational protein hydroxylation in human tissues ex vivo

A highly sensitive, specific and tissue-independent method is described to evaluate oxidative stress-mediated protein hydroxylation. in red blood cell s, frontal cortex, and liver by HPLC separation and electrochemical detecti on of protein-bound 3,4-dihydroxy-phenylalanine (DOPA) following gas-phase an-Lino acid hydrolysis of tissue protein extracts containing exclusively p roteins larger than 3 kDa. Simultaneous measurement of protein tyrosine (Ty r) content using fluorescence detection results in a tissue specific DOPA/T yr ratio that may reflect oxidative stress-mediated protein modifications i n disease, or following the exposure to oxidative stress-inducing agents.